Hollow silicon microneedle fabrication using advanced plasma etch technologies for applications in transdermal drug delivery

A novel production process flow is presented here for the manufacture of hollow silicon microneedles using deep reactive-ion etching (DRIE) technology. The patent-pending three-step process flow has been developed to produce multiple arrays of sharp-tipped, hollow microneedles, which facilitate easy insertion and controlled fluid injection into excised skin samples. A bevelled tip and vertical sidewalls for the microneedle have been achieved with good uniformity, despite >45% open etch area. Processing steps and etch challenges are discussed, and preliminary skin testing results are presented, showing effective needle insertion and delivery of fluorescent dye into ex vivo skin from human breast tissue

Multiple effects of silymarin on the hepatitis C virus lifecycle

Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV-infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell-to-cell spread of virus. CONCLUSION: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell

Transdermal Blood Sampling for C-peptide Is a Minimally Invasive, Reliable Alternative to Venous Sampling in Children and Adults With Type 1 Diabetes

OBJECTIVE:C-peptide and islet autoantibodies are key type 1 diabetes biomarkers, typically requiring venous sampling, which limits their utility. We assessed transdermal capillary blood (TCB) collection as a practical alternative.RESEARCH DESIGN AND METHODS:Ninety-one individuals (71 with type 1 diabetes, 20 controls; individuals with type 1 diabetes: aged median 14.8 years [interquartile range (IQR) 9.1–17.1], diabetes duration 4.0 years [1.5–7.7]; controls: 42.2 years [38.0–52.1]) underwent contemporaneous venous and TCB sampling for measurement of plasma C-peptide. Participants with type 1 diabetes also provided venous serum and plasma, and TCB plasma for measurement of autoantibodies to glutamate decarboxylase, islet antigen-2, and zinc transporter 8. The ability of TCB plasma to detect significant endogenous insulin secretion (venous C-peptide ≥200 pmol/L) was compared along with agreement in levels, using Bland-Altman. Venous serum was compared with venous and TCB plasma for detection of autoantibodies, using established thresholds. Acceptability was assessed by age-appropriate questionnaire.RESULTS:Transdermal sampling took a mean of 2.35 min (SD 1.49). Median sample volume was 50 µL (IQR 40–50) with 3 of 91 (3.3%) failures, and 13 of 88 (14.7%) <35 µL. TCB C-peptide showed good agreement with venous plasma (mean venous ln[C-peptide] – TCB ln[C-peptide] = 0.008, 95% CI [−0.23, 0.29], with 100% [36 of 36] sensitivity/100% [50 of 50] specificity to detect venous C-peptide ≥200 pmol/L). Where venous serum in multiple autoantibody positive TCB plasma agreed in 22 of 32 (sensitivity 69%), comparative specificity was 35 of 36 (97%). TCB was preferred to venous sampling (type 1 diabetes: 63% vs. 7%; 30% undecided).CONCLUSIONS:Transdermal capillary testing for C-peptide is a sensitive, specific, and acceptable alternative to venous sampling; TCB sampling for islet autoantibodies needs further assessment

Development of a novel antimicrobial-releasing glass ionomer cement functionalized with chlorhexidine hexametaphosphate nanoparticles

BACKGROUND: Glass ionomer cements (GICs) are a class of dental biomaterials. They have a wide range of uses including permanent restorations (fillings), cavity linings, fissure sealants and adhesives. One of the most common reasons for replacing a dental restoration is recurrent bacterial tooth decay around the margins of the biomaterial. Therefore, a dental biomaterial which creates a sustained antimicrobial environment around the restoration would be of considerable clinical benefit. In this manuscript, the formulation of a GIC containing novel antimicrobial nanoparticles composed of chlorhexidine hexametaphosphate at 1, 2, 5, 10 and 20% powder substitution by mass is reported. The aim is to create GICs which contain chlorhexidine-hexametaphosphate nanoparticles and characterize the nanoparticle size, morphology and charge and the release of chlorhexidine and fluoride, tensile strength and morphology of the GICs. RESULTS: The GICs released chlorhexidine, which is a broad spectrum antimicrobial agent effective against a wide range of oral bacteria, over the duration of the experiment in a dose-dependent manner. This was not at the expense of other properties; fluoride release was not significantly affected by the substitution of antimicrobial nanoparticles in most formulations and internal structure appeared unaffected up to and including 10% substitution. Diametral tensile strength decreased numerically with substitutions of 10 and 20% nanoparticles but this difference was not statistically significant. CONCLUSION: A series of GICs functionalized with chlorhexidine-hexametaphosphate nanoparticles were created for the first time. These released chlorhexidine in a dose-dependent manner. These materials may find application in the development of a new generation of antimicrobial dental nanomaterials

Natural Biomaterials for Cardiac Tissue Engineering: A Highly Biocompatible Solution.

Cardiovascular diseases (CVD) constitute a major fraction of the current major global diseases and lead to about 30% of the deaths, i.e., 17.9 million deaths per year. CVD include coronary artery disease (CAD), myocardial infarction (MI), arrhythmias, heart failure, heart valve diseases, congenital heart disease, and cardiomyopathy. Cardiac Tissue Engineering (CTE) aims to address these conditions, the overall goal being the efficient regeneration of diseased cardiac tissue using an ideal combination of biomaterials and cells. Various cells have thus far been utilized in pre-clinical studies for CTE. These include adult stem cell populations (mesenchymal stem cells) and pluripotent stem cells (including autologous human induced pluripotent stem cells or allogenic human embryonic stem cells) with the latter undergoing differentiation to form functional cardiac cells. The ideal biomaterial for cardiac tissue engineering needs to have suitable material properties with the ability to support efficient attachment, growth, and differentiation of the cardiac cells, leading to the formation of functional cardiac tissue. In this review, we have focused on the use of biomaterials of natural origin for CTE. Natural biomaterials are generally known to be highly biocompatible and in addition are sustainable in nature. We have focused on those that have been widely explored in CTE and describe the original work and the current state of art. These include fibrinogen (in the context of Engineered Heart Tissue, EHT), collagen, alginate, silk, and Polyhydroxyalkanoates (PHAs). Amongst these, fibrinogen, collagen, alginate, and silk are isolated from natural sources whereas PHAs are produced via bacterial fermentation. Overall, these biomaterials have proven to be highly promising, displaying robust biocompatibility and, when combined with cells, an ability to enhance post-MI cardiac function in pre-clinical models. As such, CTE has great potential for future clinical solutions and hence can lead to a considerable reduction in mortality rates due to CVD

Design, fabrication, and characterisation of a silicon microneedle array for transdermal therapeutic delivery using a single step wet etch process

The fabrication of silicon in-plane microneedle arrays from a simple single wet etch step is presented. The characteristic 54.7° sidewall etch angle obtained via KOH etching of (1 0 0) orientation silicon wafers has been used to create a novel microneedle design. The KOH simultaneously etches both the front and back sides of the wafer to produce V shaped grooves, that intersect to form a sharp pyramidal six-sided microneedle tip. This method allows fabrication of solid microneedles with different geometries to determine the optimal microneedle length and width for effective penetration and minimally invasive drug delivery. A modified grooved microneedle design can also be used to create a hollow microneedle, via bonding of two grooved microneedles together, creating an enclosed hollow channel. The microneedle arrays developed, effectively penetrate the skin without significant indentation, thereby enabling effective delivery of active ingredients via either a poke and patch application using solid microneedles or direct injection using hollow microneedles. This simple, scalable and cost effective method utilises KOH to etch the silicon wafer in-plane, allowing microneedles with variable length of several mm to be fabricated, as opposed to out-of-plane MNs, which are geometrically restricted to dimensions less than the thickness of the wafer. These microneedle arrays have been used to demonstrate effective delivery of insulin and hyaluronic acid into the skin

Discovery and Annotation of Two Phages that Infect Microbacterium foliorum: Tedro and Bajuniper

We isolated and purified Tedro and BAjuniper which infect the host Microbacterium foliorium. Tedro is a lytic, cluster EF phage isolated from soil collected in Hawarden, Iowa. Its genome is 56,197 bp long, circularly permuted, and includes 83 protein-coding genes and no tRNA genes. We are examining two of Tedro’s genes, genes 56 and 57, both of which are predicted to encode a DnaE-like DNA polymerase III (alpha) in more detail. Tedro_57 is twice as large as Tedro_56 so we are using additional bioinformatic tools to understand these genes. BAjuniper was isolated from soil collected in a garden in Orange City, Iowa. Its genome is 41,985 bp long. It was assigned to cluster EB. BAjuniper’s genome includes one tRNA gene and we will finalize BAjuniper’s annotation shortly

Haplotype tagging reveals parallel formation of hybrid races in two butterfly species.

Genetic variation segregates as linked sets of variants or haplotypes. Haplotypes and linkage are central to genetics and underpin virtually all genetic and selection analysis. Yet, genomic data often omit haplotype information due to constraints in sequencing technologies. Here, we present "haplotagging," a simple, low-cost linked-read sequencing technique that allows sequencing of hundreds of individuals while retaining linkage information. We apply haplotagging to construct megabase-size haplotypes for over 600 individual butterflies (Heliconius erato and H. melpomene), which form overlapping hybrid zones across an elevational gradient in Ecuador. Haplotagging identifies loci controlling distinctive high- and lowland wing color patterns. Divergent haplotypes are found at the same major loci in both species, while chromosome rearrangements show no parallelism. Remarkably, in both species, the geographic clines for the major wing-pattern loci are displaced by 18 km, leading to the rise of a novel hybrid morph in the center of the hybrid zone. We propose that shared warning signaling (Müllerian mimicry) may couple the cline shifts seen in both species and facilitate the parallel coemergence of a novel hybrid morph in both comimetic species. Our results show the power of efficient haplotyping methods when combined with large-scale sequencing data from natural populations

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient

COVAC1 phase 2a expanded safety and immunogenicity study of a self-amplifying RNA vaccine against SARS-CoV-2

BACKGROUND: Lipid nanoparticle (LNP) encapsulated self-amplifying RNA (saRNA) is well tolerated and immunogenic in SARS-CoV-2 seronegative and seropositive individuals aged 18-75. METHODS: A phase 2a expanded safety and immunogenicity study of a saRNA SARS-CoV-2 vaccine candidate LNP-nCoVsaRNA, was conducted at participating centres in the UK between 10th August 2020 and 30th July 2021. Participants received 1 μg then 10 μg of LNP-nCoVsaRNA, ∼14 weeks apart. Solicited adverse events (AEs) were collected for one week post-each vaccine, and unsolicited AEs throughout. Binding and neutralisating anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spike (S) IgG ELISA, and SARS-CoV-2 pseudoneutralisation assay. (The trial is registered: ISRCTN17072692, EudraCT 2020-001646-20). FINDINGS: 216 healthy individuals (median age 51 years) received 1.0 μg followed by 10.0 μg of the vaccine. 28/216 participants were either known to have previous SARS-CoV2 infection and/or were positive for anti-Spike (S) IgG at baseline. Reactogenicity was as expected based on the reactions following licensed COVID-19 vaccines, and there were no serious AEs related to vaccination. 80% of baseline SARS-CoV-2 naïve individuals (147/183) seroconverted two weeks post second immunization, irrespective of age (18-75); 56% (102/183) had detectable neutralising antibodies. Almost all (28/31) SARS-CoV-2 positive individuals had increased S IgG binding antibodies following their first 1.0 μg dose with a ≥0.5log10 increase in 71% (22/31). INTERPRETATION: Encapsulated saRNA was well tolerated and immunogenic in adults aged 18-75 years. Seroconversion rates in antigen naïve were higher than those reported in our dose-ranging study. Further work is required to determine if this difference is related to a longer dosing interval (14 vs. 4 weeks) or dosing with 1.0 μg followed by 10.0 μg. Boosting of S IgG antibodies was observed with a single 1.0 μg injection in those with pre-existing immune responses. FUNDING: Grants and gifts from the Medical Research Council UKRI (MC_PC_19076), the National Institute for Health Research/Vaccine Task Force, Partners of Citadel and Citadel Securities, Sir Joseph Hotung Charitable Settlement, Jon Moulton Charity Trust, Pierre Andurand, and Restore the Earth